Method of increasing expression of HLA, cell surface and TAA antigens of cells using 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol

ABSTRACT

A composition for the upregulation of expression of cell antigens, without inducing shedding, which comprises a protein kinase C activator is provided by this invention. Further provided by this invention is a method of detecting and treating tumor cells comprising contacting tumor cells with an effective amount of a protein kinase C activator for the upregulation of expression of antigens of tumor cells, without inducing antigen shedding, and detecting the presence of said antigen or then further contacting said tumor cells with an effective amount of an antibody directed to said antigen.

The invention described herein was made in the course of work underGrant Nos. CA 35675 and CA 43208 from the National Institute ofHealth-National Cancer Institute. The United States Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byArabic number within brackets. Full citations for these publications maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed therein.

The expression of both histocompatibility antigens and tumor associatedantigens of tumor cells can be augmented by treatment with bioresponsemodulators, such as interferon and tumor necrosis factor-α, and phorbolester tumor promoters, such as TPA 1,13,14,16,18,22,23,27,28!.Upregulation of additional cellular antigens can be induced to a similarextent in both normal and tumor cells by bioresponse modulatorsindicating that this effect is a general property of these compounds andnot restricted to TAAs or cells of a specific histotype (for review see1,23!). For example, various interferons have been shown to enhance theexpression of histocompatibility antigens, cellular antigens and TAAs inbreast carcinoma, central nervous system tumors, colon carcinoma andmelanoma cells 21-23,27,28,31,38!. In addition, when administered toanimals containing human tumor xenografts, recombinant human interferonaugments the ability of excised tumors to bind monoclonal antibodiesspecific for TAAs 13,21,36,42!. The use of bioresponse modifiers forincreasing the expression of TAAs by tumor cells may prove useful inreducing the antigenic heterogeneity in tumors in vivo and augmentingthe ability of monoclonal antibodies to bind to tumors (for review see1,23,25,26!).

TPA and recombinant human leukocyte (IFN-α), fibroblast (IFN-β) andimmune (IFN-γ) interferons increase both the expression and shedding ofthe tumor associated antigen BCA 225 by T47D cells and MCF-7 humanbreast carcinoma cells 36!. These compounds also increase the expressionof the TAA carcinoembryonic antigen (CEA) and HLA Class II-DR antigen inboth T47D and MCF-7 cells 20,36!. The mechanism by which TPA induces itsdiversity of effects in target cells is believed to be mediatedinitially by its binding to the Ca²⁺ -activated andphospholipid-dependent enzyme PKC which is the high affinity receptorfor TPA (for review see 10,43,44!. As a consequence of activation of PKCmany important biochemical processes are initiated in target cells,including both positive and negative feedback controls in signaltransduction pathways (for review see 43,44!. Recent studies haveimplicated PKC activation in mediating both antiviral activity andspecific gene regulatory changes induced by IFN-α, IFN-β, and IFN-γ6,8,37,46,47,50! and for review see 9!. The purpose of the present studywas to explore the possible relationship between PKC activation andantigen upregulation induced by phorbol esters and interferon. With thisaim in mind we have determined the effect of the synthetic PKC-activatorADMB, the natural PKC activators TPA and MEZ and the combination ofPKC-activators and the PKC-inhibitor H-7 on the antigenic phenotype ofT47D cells. To determine if similar biochemical pathways are involved inthe ability of IFN-β and IFN-γ to alter the antigenic phenotype of T47Dcells, we have also evaluated the effect of H-7 on interferonupregulation of the same antigens in T47D cells.

The effect of a synthetic protein kinase C (PKC) activator3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB) andthe natural PKC-activating tumor promoting agents12-0-tetradecanoyl-phorbol-13-acetate (TPA) and mezerein (MEZ) on theantigenic phenotype of carcinoma cells was studied. All three agentsincreased the surface expression of the tumor associated antigen such asBCA 225 and also of various cellular antigens, including HLA Class IIantigens, intercellular adhesion molecule-1 (ICAM-1) and c-cerbB-2.Expression of the same antigens was also upregulated to various extentin T47D cells by recombinant fibroblast (IFN-β) and immune (IFN-γ)interferon. Shedding of BCA 225 from T47D cells was induced by TPA, MEZ,IFN-β and IFN-γ, whereas ADMB did not display this activity. The abilityof ADMB, TPA and MEZ to modulate the antigenic phenotype of T47D cellsappears to involve a PKC-mediated pathway, since the PKC inhibitor, H-7,eliminates antigenic modulation. In contrast, the ability of IFN-β andIFN-γ to enhance HLA Class II antigens, c-erbB-2 and ICAM-1 expression,was either unchanged or modestly reduced by simultaneous exposure toH-7. Analysis of steady-state mRNA levels for HLA Class I antigens, HLAClass II-DRβ antigen, ICAM-1, and c-erbB-2 indicated that the ability ofH-7 to inhibit expression of these antigens in TPA-, MEZ-, andADMB-treated cells was not a consequence of a reduction in thesteady-state levels of mRNAs for these antigens. The results of thepresent investigation indicate that the biochemical pathways mediatingenhanced antigenic expression in T47D cells induced by TPA, MEZ and thesynthetic PKC activator ADMB are different than those induced byrecombinant interferons. Furthermore, upregulation of antigenicexpression in T47D cells can occur by both a PKC-dependent or aPKC-independent pathway.

SUMMARY OF THE INVENTION

This invention provided a composition for the upregulation of expressionof all antigens without inducing shedding which comprises a proteinkinase C activation for the upregulation of expression of a cell antigenwithout inducing shedding of said antigen from the cell.

Further provided by this invention is a method for decreasing tumor cellheterogeneity by upregulating the expression of an antigen withoutinducing shedding of said antigen which comprises administering anamount of a protein kinase C activator to cells to induce upregulationof expression of an antigen without inducing shedding of said antigen.

Additionally, this invention provides a method of detecting tumor cellscomprising contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells, without inducing shedding, and detecting the presence ofsaid antigen.

A method for treating tumor cells is also provided. This methodcomprises contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells without inducing antigen shedding and then contacting saidtumor cells with an effective amount of an antibody directed to saidantigen.

This invention also provides a pharmaceutical composition forupregulating the expression of antigens without inducing antigenshedding which comprises a pharmaceutically acceptable carrier and aneffective amount of a protein kinase C activator.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: Effect of H-7, TPA, MEZ and ADMB on T47D cell growthand DNA synthesis. Cell growth assays are presented in FIG. 1A. T47Dcells were seeded at 1×10⁴ cells/35 mm tissue culture plate, the mediumwas changed with the indicated compounds 24 hr later and cell numberswere determined after an additional 72 hr growth at 37° C. by CoulterCounter. Results are the average of triplicate samples/experimentalcondition which varied by ≦10%. DNA synthesis assays are presented inFIG. 1B. T47 D cells were seeded at 1.25×10⁴ cells in 0.2 ml of media in96 microtiter plates. Every 24 hr, cultures received 1 μCi of methyl-³H-thymidine and 8 hr later cells were harvested and TCA precipitablecounts were determined. Results are the average from replicate samplesexposed to the indicated compounds for 72 hr. Replicate samples variedby ≦10% and replicate studies varied by ≦15%. Further details can befound in "Detailed Description Of The Invention".

FIGS. 2A and 2B: Effect of H-7 on the upregulation of HLA Class IIantigens and c-erbB-2 antigen expression in T47D cells induced by ADMB,TPA and MEZ, T47D cells were grown for 72 hr in the presence of 0.1μg/ml of ADMB, TPA or MEZ, used alone or in combination with 0.1 μg/mlof H-7. Cell surface antigen expression was then determined by FACSanalysis as described in "Detailed Description Of The Invention."Base-line control antigen expression is given the value of 1.0. Thevalues presented are the fold-change, which represents the ratio of theexperimental MFI value versus the control MFI value for the specificantigen tested, in fluorescence in experimental versus control samples.The results presented are from a single experiment employing replicatesamples. Similar results ≦15% have been obtained in two additionalstudies. Further details can be found in "Detailed Description Of TheInvention."

FIG. 3: Effect of interferon, ADMB, TPA and MEZ, alone and incombination with H-7, on steady-state mRNA levels of HLA Class Iantigens, HLA Class II-DR.sub.β antigen, c-erbB-2, ICAM-1 and GAPDH inT47D human breast carcinoma cells. T47D cells were grown for 72 hr inthe absence (control) or presence of 500 units/ml of IFN-α or IFN-β, 50units/ml of IFN-γ, 0.1 μg/ml of TPA, MEZ or ADMB. Cultures were alsogrown in the presence of 0.1 μg/ml of H-7 for 72 hr with and without theadditional compounds indicated above. Total cytoplasmic RNA was isolatedand processed as described in "Detailed Description Of The Invention."Control: CON; recombinant human leukocyte interferon-A: IFN-αA;recombinant human fibroblast interferon: IFN-β; recombinant human immuneinterferon: IFN-γ; ADMB;3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol; TPA;12-0-tetradecanoyl-phorbol-13-acetate; MEZ; mezerein;H-7:(1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition for the upregulation of expressionof cell antigens without inducing shedding which comprises a proteinkinase C activator for the upregulation of expression of a cell antigenwithout inducing shedding of said antigen from the cell. In oneembodiment of the invention the proein kinase C actovator is a syntheticprotein kinase C activator. In the preferred embodiment of the inventionthe protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB).

In an embodiment of the invention the cell antigen is a tumor associatedantigen (TAA). In yet another embodiment of the invention the cellantigen is a cell surface antigen. The cell antigen may also be ahistocompatibility antigen. Examples of tumor associated antigens thatcan be upregulated by this invention include, but are not limited to,BCA 225, c-erb B2, or carcinoembryonic antigen. In one preferredembodiment of the invention the cell surface antigen is intercellularadhesion molecule-1.

The tumor of the tumor associated antigen may be, but is not limited to,breast carcinoma or colon carcinoma.

Further provided by this invention is a method for decreasing tumor cellheterogeneity by upregulating the expression of an antigen withoutinducing shedding of said antigen which comprises administering anamount of a protein kinase C activator to cells to induce upregulationof expression of an antigen without inducing shedding of said antigen.In one embodiment of the invention the protein kinase C activator is asynthetic protein kinase C activator. In a preferred embodiment of theinvention the synthetic protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB). Whenusing ADMB for decreasing tumor cell heterogeneity by upregulating theexpression of an antigen, without inducing shedding, the effectiveamount is from about 0.01 μg/ml to about 10 μg/ml.

In one embodiment of the invention the antigen is a tumor associatedantigen. In another embodiment of the invention the antigen is ahistocompatibility antigen. In yet another embodiment of the inventionthe antigen is a cell surface antigen. The tumor of the tumor associatedantigen can be, but is not limited to, breast carcinoma or coloncarcinoma. In certain embodiments of the invention the tumor associatedantigen is BCA 225. In other embodiments of the invention the tumorassociated antigen is carcinoembryonic antigen. In yet anotherembodiment of the invention the tumor associated antigen is c-erb B2. Inanother embodiment of the invention the cell surface antigen inintercellular adhesion molecule-1.

This invention also provides a method of detecting tumor cellscomprising contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells, without inducing antigen shedding, and detecting thepresence of said antigen. In one embodiment of the invention the proteinkinase C activator is a synthetic protein kinase C activator. In thepreferred embodiment of the invention the synthetic protein kinase Cactivator is 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol(ADMB). When using ADMB in the subject invention the effective amount isfrom about 0.01 μg/ml to about 10 μg/ml. The tumor of the tumorassociated antigen may be, but is not limited to, breast carcinoma orcolon carcinoma. In certain embodiments of the invention the tumorassociated antigen is BCA 225. The tumor associated antigen can also becarcinoembryonic antigen or c-erb B2. In yet another embodiment of theinvention the antigen is intercellular adhesion molecule-1.

Further provided by this invention is a method of treating tumor cellscomprising contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells without inducing antigen shedding and then contacting saidtumor cells with an effective amount of an antibody directed to saidantigen. In one embodiment of the invention the protein kinase Cactivator is a synthetic protein kinase C activator. In the preferredembodiment of the invention the synthetic protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB). Whenusing ADMB, the effective amount is from about 0.1 μg/ml to about 10μg/ml. The antigen may be, but is not limited to, a tumor associatedantigen or a cell surface antigen. The tumor cells may be, but are notlimited to, breast carcinoma or colon carcinoma. In preferredembodiments of the invention the tumor associated antigen is BCA 225,carcinoembryonic antigen, or c-erb B2. In yet another embodiment of theinvenition the cell surfade antigen is intercellular adhesionmolecule-1.

Additionally, this invention provides a pharmaceutical composition forupregulating the expression of antigens without inducing antigenshedding which comprises a pharmaceutically acceptable carrier and aneffective amount of a protein kinase C activator. In one embodiment ofthe invention the protein kinase C activator is a synthetic proteinkinase C activator. In the preferred embodiment of the invention thesynthetic protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB). Whenthe protein kinase C activator is ADMB, the effective amount is fromabout 0.01 μg/ml to about 10 μg/ml. The antigen may be, but is notlimited to being, a tumor associated antigen, a cell surface antigen, orhistocompatibility antigen. The tumor of the tumor associated antigenmay be, but is not limited to, breast carcinoma or colon carcinoma. Inone embodiment of the invention the tumor associated antigen is BCA 225.In another embodiment of the invention the tumor associated antigen iscarcinoembryonic antigen. In yet another embodiment of the invention thetumor associated antigen is c-erb B2. In one embodiment of the inventionthe cell surface antigen is intercellular adhesion molecule-1.

For the purposes of this invention, "physiologically acceptable carrier"means any of the standard pharmaceutical carriers. Examples of suitablecarriers are wellknown in the art and may include, but are not limitedto, any of the standard pharmaceutical carriers.

Methods of determining the effective amounts are wellknown in the art. Aperson of ordinary skill in the art can easily extrapolate the effectiveamounts as determined in vitro, and apply it to living organisms todetermine the effective concentrations in vivo.

MATERIALS AND METHODS Example 1

Cell Cultures

The T47D clone 11 human breast carcinoma cell line 32! was grown in RPMI1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate,fungizone (0.25 μg/ml), streptomycin (50 μg/ml), penicillin (50 U/ml),10% fetal bovine serum (FBS), β-estradiol and insulin (0.1 μu/ml) at 37°C. in a 5% CO₂ /95% air humidified incubator. Cultures were maintainedin the logarithmic phase of growth by subculturing at a 1:5 or 1:10split-ratio when cells approached confluency.

Example 2

Growth and ³ H-Thymidine Incorporation Assays

T47D cells were seeded at 5×10³ cells/ml in 35 mm tissue culture platesand 24 hr later the medium was changed with the indicated compounds.Seventy-two hr later the cells were resuspended in trysin/versene(0.125%/0.02%, w/w) and counted using a Z_(M) Coulter Counter (CoulterElectronics). For ³ H-thymidine incorporation studies, T47D cells wereseeded at 1.25×10⁴ cells in 0.2 ml of media in 96 well microtiterplates. Every 24 hr, plates received 1 μCi of methyl-³ H-thymidine(specific activity 10 μCi/mmol) (ICN Radiochemicals, Irvine, Calif.),cells were harvested 8 hr after the addition of labeled thymidine andTCA precipitable counts 55! were determined using a Packardscintillation counter. Replicate samples varied by ≦10% and replicatestudies varied by ≦15%.

Example 3

Monoclonal Antibodies

IgG₁ murine monoclonal antibody Cu18 and Cu46 recognize two differentepitopes of a highly restricted breast carcinoma associated glycoproteinof M_(r) 225,000 to 250,000 (BCA 225) 41!. This TAA is expressedintracytoplasmically and on the membrane of 94% of breast tumors testedand in the T47D human breast carcinoma cell line. BCA 225 is shed intothe culture medium by T47D cells and into the sera of breast cancerpatients. IgG_(2a) monoclonal antibody L243 (anti HLA Class II-DR)recognizes a monomorphic HLA Class II-DR-α epitope (ATCC M355).Monoclonal antibody CL203.4 40!, which recognizes ICAM-I, was kindlyprovided by Dr. S. Ferrone, New York Medical College, NY. The c-erbB-2monoclonal antibody which recognizes the extracellular domain ofc-erbB-2 was obtained from Triton Biosciences, Inc., Alamedia, Calif.Monoclonal antibodies Cu18 and Cu46 were used at 0.05 μg/ml andmonoclonal antibodies interacting with HLA Class II-DR, ICAM-1 andc-erbB-2 were used at 10 μg/ml. For each experiment, isotype matchedcontrol backgrounds (IgG for Cu18, Cu46 and ICAM-1, IgG_(2a) for HLAClass II and IgG_(2b) for c-erbB-2) were subtracted from theexperimental results. Background from antimouse IgG FITC antibody wasalso subtracted from experimental results. We never observed backgroundshigher than 2% of total cells for isotypic mouse IgG or higher than 5%for antimouse IgG FITC.

Example 4

Analysis of TRA and Cellular Antigen Expression by FluorescenceActivated Cell Sorter (FACS) Analysis

T47D cells treated with the various compounds were analyzed by flowcytometry using appropriate monoclonal antibody concentrations asdescribed previously 36!. Briefly, 1×10⁵ cells were incubated with thetest antibody for 30 min at 4° C., washed 3× with PBS and incubated witha goat α mouse F (ab)₂ FITC conjugated test antibody at a 1:40 dilutionfor 30 min at 4° C. Cells were then washed 3× with PBS and analyzed on aFACStar (Beckon Dickinson, Mountain View, Calif.) or a Coulter Epics IVFACS (Coulter, Hialeah, Fla.). Results are expressed as meanfluorescence intensity (MFI) units which were determined as describedpreviously 36!. MFI=(mean channel fluorescence positive antibody-bindingcells)-(mean channel fluorescence in unstained cells×% of fluorescencepositive cells in the unstained population). All studies were performeda minimum of three or four times with duplicate samples in eachexperiment. Replicate samples within individual experiments varied by≦10% and variation between experiments were generally ≦20%.

Example 5

Analysis of the Synthesis and Shedding of BCA 225

After appropriate incubation times with the various compounds celllysates were prepared from T47D cells. Cells were washed 3× in PBS, pH7.6, pelleted and incubated for 1 hr at room temperature in 20 mM TrisHCl, pH 7.4, with PMSF. Cultures were then homogenized with a Teflonhomogenizer, centrifuged at 3000 RPM for 10 min at 4° C. and thesupernatant was mized 1:2 with 20 mM Tris-HCl, pH 7.4, containing 0.5%NP40 (Sigma). After 1 hr at 4° C., the mixture was spun at 3000 RPM for10 min and the supernatant was passed through an EXTRACTIGEL column(Pierce, Ill.) to remove excess detergent. Protein concentration wasdetermined by the BCA micro-method (Pierce, Ill.). BCA 225 levels incell extracts and supernatants from control and treated T47D cells werequantitated using a double-determinant ELISA assay 4,36!. Briefly, Cu18monoclonal antibody coated Nunc Immunoplates (Nunc, Denmark) wereblocked with 1% BSA (Sigma, RIA Reagent Grade) and incubated with a 1:2dilution of supernatant in duplicate. Standard values for a partiallypurified BCA 225 preparation were used at a range of 0 to 10 μg/ml inRPMI 1640 plus 10% FBS (T47D growth medium). After hp incubation andthree washings with PBS 0.1% Tween 20 a Cu46 monoclonal antibodyconjugated to peroxidase was applied to the plate, incubated for 2 hr,washed 6× in PBS 0.1% Tween 20 and the reaction was developed with 16 ngOPD (Sigma, St. Louis) and 4 μl of 30% H₂ O₂ in McIlvans buffer, pH 9.6.The plates were read on a Dynatech Elisa reader and a linear standardcurve was generated and used to calculate the relative amount of BCA 225in cell lysates and shed into the culture medium. Results were adjustedto ng BCA 225 per mg of protein, or per 1×10⁶ cells. Replicate samplesvaried by ≦10% and replicate experiments varied by ≦20%.

Example 6

RNA Isolation and Northern Blotting Analysis

Steady-state levels of HLA Class I, HLA Class II-DR.sub.β, c-erbB-2 andICAM-I mRNA in control and treated T47D cells were determined byNorthern blotting analysis of total cytoplasmic RNA probed withappropriate ³² P-labeled gene probes as previously described 3,56!.Northern blots were also probed with a ³² P-labeled glyceraldehydephosphate dehydrogenase (GAPDH) 12,54! gene probe to verify equal mRNAexpression under various experimental conditions. Followinghybridization, the filters were washed and exposed for autoradiography.Radioautograms were analyzed by densitometer to determine fold-change inmRNA expression as a result of treatment with the different antigenicmodulating agents, with or without cocultivation with H-7.

Example 7

Reagents

Recombinant human leukocyte (IFN-αA) and immune (IFN-γ) interferons wereproduced in Escherichia coli and purified to homogeneity as previouslydescribed. 19,49,53!. These interferons were kindly provided by Dr.Sidney Pestka, UMDNJ-Robert Wood Johnson Medical School, Piscataway,N.J. Recombinant human fibroblast (IFN-β) interferon, with a serinesubstituted for a cysteine at position 17 of the molecule 39!, wassupplied by Triton Biosciences Inc., Alameda, Calif. as a lycophilizedpowder with a concentration of 4.5×10⁷ units/ml. The interferon titerswere determined by a cytopathic effect inhibition assay with vesicularstomatitis virus on a bovine kidney cell line (MDBK) or human fibroblastAG-1732 cells 49!. Concentrated stocks of interferons were aliquotes,frozen at -80° C., thawed immediately prior to use and diluted to theappropriate concentrations in DMEM supplemented with 5 or 10% FBS. TPA(12-0-tetradecanoyl-phorbol-13-acetate), MEZ (mezerein), and ADMB(3-(N-Acetylamino)-5-(N-Decyl-N-Methylamino)-benzyl alcohol) wereobtained from LC Services Corp., Woburn, Mass. Stock solutions of 1mg/ml (TPA and MEZ) or 10 mg/ml (ADMB) were prepared indimethylsulfoxide, aliquoted and stored at 20° C. The PKC inhibitor H-7(1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) (Hidakaet al., 1984) (Sigma) was prepared in distilled H₂ O and stored at 4° C.For experiments, aliquots were thawed and dispensed in Dulbecco'smodified Eagle's medium (DMEM) containing 5 or 10% FBS to yieldappropriate final concentrations. The solvent DMSO at (0.025 to 0.05%)did not alter the growth or antigenic expression of T47D cells.

RESULTS Example 8

Effect of TPA, MEZ and ADMB on Growth and DNA Synthesis in T47D Cells

In preliminary studies, the dose-range and time-course of induction ofantigenic enhancement in T47D cells by TPA, MEZ and ADMB, as well as theeffect of varying doses of H-7 on this process, was determined (data notshown). These studies indicated that the optimum effect on antigenicexpression in T47D cells exposed to TPA, MEZ or ADMB occurred by 72 hr.The most effective dose of TPA, MEZ or ADMB inducing upregulation of BCA225, HLA Class II antigens, ICAM-1 and c-erbB-2 in T47D cells was foundto be 0.1 μg/ml. The ability of these PKC activators to induce increasedantigenic expression in T47D cells was inhibited by the simultaneousexposure to 0.1 μg/ml of the PKC inhibitor H-7 (Tables 1 and 2). Theeffect of TPA, MEZ, ADMB and H-7 on 72 hr growth and ³ H-thymidineincorporation in T47D cells is shown in FIGS. 1A and 1B. When exposed to0.1 μg/ml of the respective compounds, growth and DNA synthesis wassuppressed to the greatest degree in TPA treated cells. In contrast, atthe same dose of 0.1 μg/ml, MEZ and ADMB only marginally altered growthand DNA synthesis in T47D cells (FIGS. 1A and 1B). No additive orsynergistic effect on 72 hr growth suppression was observed when TPA orMEZ were used in combination with 0.1 μg/ml of H-7 (data not shown).

Example 9

Effect of TPA, MEZ and ADMB, Alone and in Combination with H-7, on theAntigenic Phenotype of T47D Cells

When tested for reactivity with specific monoclonal antibodies, controlT47D cells displayed the following constitutive antigenic phenotype: 10to 20% of cells (with a mean channel fluorescence of 180 to 210) werepositive for HLA Class II antigen (HLA-DR) expression; 5 to 10% (with amean channel fluorescence of 110-130) were positive for c-erbB-2expression; 80 to 90% (with a mean channel fluorescence of 170 to 200)were positive for ICAM-1 expression; 85 to 95% (with a mean channelfluorescence of 180 to 210) were positive for the TAA BCA 225 (asmonitored by the monoclonal antibody Cu18); and 60 to 70% (with a meanchannel fluorescence of 140 to 170) were positive for BCA 225 (asmonitored by the monoclonal antibody Cu46). The effect of TPA and MEZ,alone and in combination with 0.1 μg/ml of H-7, on HLA Class IIantigens, c-erbB-2 and ICAM-1 expression in T47D cells is shown inTable 1. H-7 did not significantly alter the de novo expression of anyof these antigens in T47D cells. However, when administered inconjunction with TPA or MEZ, H-7 effectively blocked the ability ofthese PKC stimulators to enhance antigenic expression. In the experimentshown in Table 1, MEZ was somewhat more effective than TPA in enhancingc-erbB-2 and ICAM-1 expression. An increased activity, at comparabledoses, of MEZ over TPA in enhancing the expression of these antigens, aswell as the TAA BCA 225, has been found in several additional studies(unpublished data and Table 2 and FIGS. 2A and 2B). For comparisonpurposes, a single experiment is shown in Table 1. In this experiment,the combination of MEZ +H-7 resulted in the lack of detectable c-erbB-2expression. This result may reflect technical difficulties rather than acomplete suppression in c-erbB-2 expression, since in additional studiesH-7 blocked the ability of MEZ to enhance the expression of this antigenwithout completely eliminating c-erbB-2 expression (unpublished data andFIGS. 2A and 2B).

Recent computer modeling studies have resulted in the synthesis ofcompounds which inhibit the binding of phorbol esters to PKC and whichactivate PKC in platelets resulting in the phosphorylation of a specific40 kDa protein substrate 58!. We have presently tested one of thesephorbol ester pharmacophores, ADMB, for its ability to upregulate thesame antigens in T47D cells previously shown to be modulated by TPA andMEZ. A comparison of the efficacy of upregulation of HLA Class IIantigens and c-erbB-2 in T47D cells exposed to TPA, MEZ and ADMB, in thepresence or absence of H-7, is presented in FIGS. 2A and 2B. In the caseof HLA Class II antigens, ADMB was somewhat more effective than TPA andMEZ in inducing upregulations, whereas H-7 reduced or eliminatedenhancement when applied in combination with these PKC activators. Inthe case of c-erbB-2, MEZ was the most effective PKC activator tested inenhancing expression and as observed with HLA Class II antigens H-7reduced this antigenic upregulation.

A series of experiments were conducted to determine the effect of TPA,MEZ and ADMB, alone and in combination with H-7, on the synthesis,surface expression and shedding of the TAA BCA 225 (Table 2). Thesynthesis of BCA 225 was increased following exposure to all of the PKCactivators, with MEZ being most effective in enhancing the synthesis ofthis TAA. Similarly, MEZ was the most effective of the three PKCactivators in enhancing the surface expression of BCA 225 in T47D cells.As observed with the other antigens analyzed, H-7 effectively blockedboth the enhanced synthesis and surface expression of BCA 225. Whencompared for their ability to induce shedding of BCA 225 from T47Dcells, a differential response was observed between the three PKCactivators (Table 2). Both MEZ and TPA enhance shedding of BCA 225, withMEZ again being more effective than TPA, whereas ADMB did not inducethis effect in T47D cells. As observed with both synthesis and shedding,H-7 reduced the ability of MEZ and TPA to induce shedding of BCA 225.

Example 10

Effect of IFN-β and IFN-γ, Alone and in Combination with H-7, on theAntigenic Phenotype of T47D Cells

We have previously demonstrated that both IFN-β and IFN-γ caneffectively enhance the expression of BCA 225, HLA Class II antigens andICAM-I expression in T47D cells 36!. Optimum enhancement was observed by72 hr with ranges of interferon of 500 to 1000 units/ml of IFN-α orIFN-β and 50 to 500 units/ml of IFN-γ, in the presence or absence of 1.0μg/ml of H-7, is shown in Tables 3 and 4. A higher dose of H-7 wasemployed in this study because the lower H-7 concentration of 0.1 μg/mldid not block enhanced antigenic expression in interferon treated T47Dcells (data not shown). IFN-γ was more effective (even at lowerconcentrations) than IFN-β in enhancing the expression of BCA225, HLAClass II antigens and ICAM-1. IFN-α also increased the expression of thethree antigens tested, but to a lower extent than IFN-β or IFN-γ (datanot shown). For example, in the experiment shown in Table 3, 500units/ml of IFN-α enhanced HLA Class II expression from an MFI of 2,814to an MFI of 11,660 (a 4-fold increase) as opposed to a 11- and a69-fold increase, respectively, in HLA Class II expression in cellsexposed to IFN-β or IFN-γ (data not shown). In a member of experiments,the de novo level of expression of specific antigens and the absolutelevel of upregulation varied. Part of this difference may reflect theuse of a different FACS with different sensitivities for determining MFIunits and/or innate differences in antigenic expression of cells as aconsequence of the cell cycle. Although this makes it difficult todirectly compare absolute levels of upregulation with the phorbol estersand ADMB versus the interferons, it still permits a comparison of theeffect of H-7 on upregulation. In experiments simultaneously comparingthe various compounds, IFN-γ was generally a more effective enhancer ofHLA Class II antigens and ICAM-1 than the other agents, whereas MEZ wasgenerally more effective in modifying c-erbB-2 and BCA 225 expression.Unlike antigenic upregulation induced by the phorbol esters and ADMBwhich was inhibited by H-7, even the higher dose of H-7 (1.0 μg/ml) didnot inhibit the ability of IFN-β or IFN-γ to enhance BCA 225, HLA ClassII antigens and ICAM-1 expression in T47D cells.

To determine if H-7 could inhibit the ability of interferon to enhancethe synthesis or shedding of BCA 225 in T47D cells, cultures were grownfor 72 hr in the presence of 500 units/ml of IFN-β or 50 units/ml ofIFN-γ and in the presence or absence of 1 μg/ml of H-7 (Table 4). As hadbeen observed for interferon enhanced expression of BCA 225, H-7 did notinhibit the synthesis or shedding of BCA 225 induced in T47D cells byinterferon. These results demonstrate that the ability of TPA and MEZversus IFN-β and IFN-γ to upregulate the synthesis, expression andshedding of BCA 225 may occur by different mechanisms.

Example 11

Effect of ADMB, TPA, MEZ and Interferon, Alone and in Combination withH-7, on the Steady-State Levels of HLA Class I Antigens, HLA ClassII-DRβ Antigen, ICAM-1 and c-erbB-2 in T47D Cells

To determine if the increase in HLA Class II-D.sub.β antigen, ICAM-1 andc-erbB-2 expression in T47D cells resulting from 72 hr ADMB, TPA, MEZ,IFN-α, IFN-β or IFN-γ treatment involved enhanced mRNA expression, thesteady-state levels of mRNA for the respective genes were determined(FIG. 3). In the case of HLA Class II-DR antigen, small increases inmRNA levels were observed in IFN-α (1.1-fold), IFN-β (1.2-fold), IFN-γ(1.25-fold) and MEZ (1.2-fold) treated T47D cells. ADMB and TPA did notincrease HLA Class II-DR.sub.β antigen, RNA expression, although ADMBwas slightly more effective than MEZ in enhancing cell surfaceexpression of this antigen in T47D cells (FIGS. 2A and 2B). MEZ andIFN-γ were the most effective enhancers of HLA Class II-DR.sub.β antigenmRNA expression, whereas IFN-γ was more effective than MEZ in enhancingexpression of this antigen in T47D cells (Tables 1 and 3). Whencotreated with the respective antigenic modulating compound and H-7,only minimal changes in HLA Class II-DR.sub.β antigen mRNA levels(<1.15-fold) were observed. These observations are in contrast to theHLA Class II-DR antigenic modulation induced by these agents in T47Dcells. As described above, H-7 effectively blocked ADMB, TPA and MEZenhancement of HLA Class II-DR antigen expression in T47D cells (Table3). In the case of HLA class I antigen expression, mRNA levels werevariably increased following treatment with IFN-α(1.9-fold), IFN-β(2.1-fold), IFN-γ(1.8-fold), TPA (1.3-fold) and MEZ (1.75-fold), whereasH-7 only marginally altered mRNA levels (≦1.2-fold) for this antigen(Table 3). With respect to surface expression, as observed with HLAClass II-DR antigen expression, H-7 inhibited upregulation induced byADMB, TPA or MEZ, but not upregulation induced by the interferons (datanot shown). These observations indicate that cell surface expressionchanges in both HLA Class I antigens and HLA Class II-DR antigen in T47Dcells is not a consequence of a reduction in mRNA levels for these geneproducts. Further studies are required, however, to determine if theantigenic modulating agents, employed alone or in combination with H-7,modulate the rate of mRNA synthesis and/or decay of mRNA synthesis forHLA Class II-DR or HLA Class I antigens in T47D cells.

ICAM-1 mRNA levels were increased a maximum of only 1.3-fold after 72 hrtreatment under the various experimental conditions and H-7 onlymodestly altered ICAM-1 expression (FIG. 3). In the case of c-erbB-2, amaximum increase of only 1.2-fold in the levels of mRNA were apparentafter 72 hr treatment with the various agents. Similarly, nodifferential change in c-erbB-2 mRNA was observed in T47D cells grown inthe presence of any of the antigenic modulating agents plus H-7. Theseresults again contrast those measuring surface expression of theseantigens. As demonstrated in Tables 1 and 3, H-7 partially inhibitedenhanced ICAM-1 expression (Table 3). Similarly, H-7 inhibited enhancedsurface expression of c-erbB-2 induced by ADMB, TPA and MEZ (FIGS. 2Aand 2B and Table 1), whereas it did not alter upregulation induced bythe interferons (data not shown). These results provide further supportfor the lack of a direct correlation between the levels of ICAM-1 andc-erbB-2mRNA and antigenic expression in cells treated with thecombination of ADMB, TPA, MEZ or interferon and H-7. Further studies arerequired, however, to determine if the antigenic modulating agents,employed alone or in combination with H-7, modulate the rate of mRNAsynthesis and/or decay of mRNA synthesis for ICAM-1 or c-erbB-2 in T47Dcells.

DISCUSSION

Among the diversity of interferon effects on target cells, recentinvestigations have focused on the ability of these bioresponsemodulators to enhance the expression of both histocompatibility antigensand TAAs in tumor cells (for review see 1,9,23,25,26!). These studiesindicate that interferon may prove valuable in altering the phenotype oftumor cells rendering them more accessible to monoclonal antibodytargeting 13,21,36,42!. A frequent observation is that interferonfunctions predominantly as an enhancer of antigenic expression, ratherthan an inducer of de novo expression of specific antigens9,15,16,25,31!. At the present time, little information is available onthe biochemical mechanisms underlying interferon upregulation ofantigenic expression. Studies comparing the protein synthesisrequirements for antigenic modulation induced by types I (IFN-α/β) andtype II (IFN-γ) interferon in human melanoma cells suggest thatdifferent biochemical pathways mediate upregulation of both majorhistocompatibility complex (MHC) and non-MHC encoded glycoproteinsinduced by these compounds 17!. IFN-γ enhancement of antigen expressiondepends on continued protein synthesis, whereas the modulatory effect ofIFN-α and IFN-β can occur in the presence of the protein synthesisinhibitor cycloheximide. Numerous studies have also indicated that typesI and II interferons can differ in their effects on TAA expression inthe same tumor cell (for review see 1,23,25,48!. Both the absolute levelof antigenic modulation induced by different interferons, as well as thetype of effect elicited in specific target cells, i.e. eitherstimulatory or inhibitory, has been shown to vary (for review see 1,23!.In the present study we have addressed the potential relationshipbetween PKC activation and antigenic modulation induced by recombinantIFN-β and IFN-γ in the human breast carcinoma cell line T47D. Since bothTPA and MEZ can augment the expression of the same histocompatibilityantigens and TAAs in T47D cells as recombinant interferons and theseagents appear to work directly via activation of PKC (for review see43,44!, we have also incorporated these agents in our studies.

The enhanced cellular antigenic expression induced in T47D cells by TPA,MEZ and ADMB was eliminated by simultaneous incubation with thePKC-inhibitor H-7. Similarly, the ability of TPA and MEZ to enhance thesynthesis and shedding of the TAABCA 225 was also blocked by H-7. Incontrast, ADMB failed to induce increased shedding of BCA 225 by T47Dcells, whereas it was similarly active as TPA in enhancing BCA 225synthesis and expression (Table 2). These observations suggest that ADMBcan differentially modify the antigenic phenotype in T47D cells incomparison with TPA and MEZ, i.e. it can augment synthesis andexpression without enhancing shedding of specific TAAs. Interestingly,ADMB was found to induce HLA Class II antigens in a similar manner asTPA and MEZ in T47D cells. HLA Class II antigens have been shown to beinvolved in the differentiation of mammary epithelium 11! and they playa critical role in antigen presentation to T-cells 35!, the transport ofkey intracellular peptides to the extracellular milieu 57! andrecruitment of lymphod cells to tumor cells 11,35,59!. The ability ofADMB to enhance both HLA Class II antigens and TAA expression on T47Dcells could have implications with respect to the induction of an immuneresponse to this tumor in vivo. A severe limitation preventing the useof TPA or MEZ as potential immunomodulators in humans is there welldocumented tumor promoting activity in the mouse skin two-stagecarcinogenesis assay for review see 51-52!. At the present time, ADMBhas not been tested for in vivo toxicity, tumor promoting activityand/or in vivo immunomodulatory properties. However, if this compoundcan pass this scrutiny, it could prove useful as an antigenic modulatingagent in situations where increased surface expression without aconcomitant increase in TAA shedding is desired.

Previous studies have indicated a possible involvement of activation ofPKC in the early events associated with IFN-α action in specific targetcells 2,6,7,46,50,60-62!. Although not studied as extensively, anassociation between PKC activity and both IFN-β- and IFN-γ-inducedcellular changes has also been suggested 8,29,30,45,47!. In addition, adifferential role for PKC in IFN-α/β versus IFN-γ induced cellular andgene expression changes in the same target cell has also been observed6,37,47!. Both IFN-β and IFN-γ produced similar antigenic changes inT47D cells as TPA and MEZ, including enhancing the shedding of BCA 225.However, up-regulation of antigen expression and increased sheddinginduced by IFN-β and IFN-γ was not inhibited by H-7 (Table IV). IFN-αwas less effective than either IFN-β or IFN-γ in modifying the antigenicphenotype of T47D cells and its activity also was not blocked by H-7(data not shown). These results suggest that the mechanism by whichinterferons modulate antigen expression in T47D cells occurs by aPKC-independent pathway. A similar dissociation between PKC activationand the ability of IFN-γ and TPA to induce specific antigenic expressionchanges in human keratinocyte cultures has recently been reported 24!.IFN-γ and TPA both enhanced ICAM-1 expression in human keratinocytecultures and the enhancement effect of TPA, but not that of IFN-γ, wasinhibited by H-7. Similarly, only IFN-γ induced HLA Class II-DR antigenexpression in human keratinocytes and H-7 also failed to block thisinduction. Koide et al. 34! demonstrated that IFN-γ induction of HLAClass II-DR antigen expression in H1-60 cells also was not modified byH-7. In contrast, W7 (a calmodulin antagonist) blocked IFN-γ-inductionof HLA Class II-DR expression in HL-60 cells supporting a possibleinvolvement of calcium/calmodulin in antigenic modulation in this cellline. Similarly, treatment of murine macrophages with IFN-γ resulted inthe induction of both increased mRNA and MHC I-A.sub.β antigenexpression, and both of these parameters were unaltered in the presenceof H-7 5!. W-7 did, however, modify the MHC I-A.sub.β antigen mRNAinduction process elicited by IFN-γ treatment in murine macrophages. Inthe case of the human melanoma cell line H0-1, the enhanced expressionof HLA Class I antigens, HLA Class II antigens and ICAM-1 induced byIFN-γ was again only marginally affected by H-7 26!. Since upregulationof antigen expression in T47D cells induced by PKC activators such asTPA, MEZ and ADMB are inhibited by H-7, whereas similar changes inducedby the interferons are not blocked by H-7, these results furtherindicate that the mechanism controlling antigenic modulation in specificcell cultures is dependent on the specific inducer employed andantigenic modulation can occur by both a PKC-independent and aPKC-dependent pathway.

The mechanism by which ADMB and MEZ versus IFN-γ enhance the expressionof specific cellular antigens and TAAs in T47D is not presently known.Analysis of steady state mRNA levels of HLA Class I, HLA ClassII-DR.sub.β, ICAM-1 and c-erbB-2 in cells treated with these differentcompounds indicated various levels of modulation which did not correlatedirectly with the relative level of change in surface expression ofthese antigens. Similarly, H-7 did not significantly alter the level ofmRNA for the various antigens under any of the experimental conditions.These results suggest that the ability of H-7 to modulate the antigenicenhancing properties of ADMB, TPA, MEZ and the interferons may occur ata posttranscriptional level. Alternatively, these agents may modifyantigenic expression by altering the rate of mRNA transcription and/ormRNA stability. In the case of BCA 225, H-7 may exert its suppressiveeffect on ADMB-, TPA-, and MEZ-induced increases in surface expressionby inhibiting the ability of these compounds to enhance the synthesis ofthis TAA in T47D cells. Alternatively, H-7 might block antigenicupregulation in ADMB, TPA and MEZ treated cells by preventing thenecessary biochemical alterations responsible for the insertion of thevarious antigens into the cell membrane in a form recognized by themonoclonal antibodies employed. Further studies are clearly required todetermine the mechanism by which specific antigenic modulatorsupregulate antigen expression and the mechanism by which H-7 selectivelyinhibits this process in cells treated with ADMB, TPA or MEZ. Thepresent model system should prove useful in determining the biochemicalmechanism(s) underlying antigenic upregulation in response to diversetransmembrane signalling agents. With this information it may bepossible in the future to design strategies and molecules specificallytailored to alter the antigenic phenotype of tumor cells making themmore accessible to monoclonal antibody targeted therapeutic approaches.

                  TABLE 1                                                         ______________________________________                                        Effect of the PKC Inhibitor H-7 on Upregulation of                            HLA Class II antigens, c-erbB-2 and ICAM-1 by TPA and MEZ in                  T47D Human Breast Carcinoma cells.                                                      Antigenic                                                           Experimental                                                                            Expression (MFI)                                                    Conditions.sup.a                                                                        HLA Class II c-erbB-2 ICAM-1                                        ______________________________________                                        Control   3,410        7,144    20,975                                        H-7       4,563        7,045    21,538                                                  .sup. (1.3).sup.b                                                                          (1.0)    (1.0)                                         TPA       9,984        16,100   63,175                                                  (2.9)        (2.3)    (3.0)                                         TPA + H-7 4,778        8,385    47,542                                                  (1.4)        (1.2)    (2.3)                                         MEZ       10,260       18,385   85,745                                                  (3.0)        (2.6)    (4.1)                                         MEZ + H-7 3,654        Not      39,486                                                  (1.1)        Detected (1.9)                                         ______________________________________                                         .sup.a T47D cells were grown for 72 hr in 0.1 μg/ml TPA or 0.1 μg/m     MEZ, in the presence of absence of 0.1 μg/ml H7. Cells were                resuspended, incubated with monoclonal antibodies specific for HLA Class      II antigens, cerbB2 or ICAM1 and antimouse FITC secondary antibody. Cells     were then analyzed by flow cytometry using a FACStar (Beckon Dickinson,       Mountain View, CA) and antigenic expression as expressed as mean              fluorescence intensity (MFI) units. Further details can be found in           "Detailed Description of the Invention.                                       .sup.b Numbers in brackets reflect the level of upregulation versus           untreated control cells (equivalent to 1.0).                             

                  TABLE 2                                                         ______________________________________                                        Effect of the PKC Inhibitor H-7 on the Enhanced                               Synthesis, Expression and Shedding of BCA 225 Induced by TPA,                 MEZ and ADMB in T47D Human Breast Carcinoma Cells.                                                              BCA 225                                                BCA 225     BCA 225    Shedding.sup.d                              Experimental                                                                             Synthesis.sup.b                                                                           Expression.sup.c                                                                         (ng/ml/10.sup.6                             Conditions.sup.a                                                                         (ng/mg protein)                                                                           (MFI)      cells)                                      ______________________________________                                        Control    164         6,426      28                                          H-7        .sup. 142 (0.9).sup.e                                                                     5,428 (0.8)                                                                              23 (0.8)                                    TPA        394 (2.4)   11,696 (1.8)                                                                             84 (3.0)                                    TPA + H-7  191 (1.2)   7,434 (1.2)                                                                              35 (1.3)                                    MEZ        507 (3.1)   16,065 (2.5)                                                                             147 (5.3)                                   MEZ + H-7  225 (1.4)   7,068 (1.1)                                                                              39 (1.4)                                    ADMB       299 (1.8)   12,825 (2.0)                                                                             26 (0.9)                                    ADMB + H-7 178 (1.1)   7,018 (1.1)                                                                              34 (1.2)                                    ______________________________________                                         .sup.a T47D cells were incubated for 72 hr in the presence of 0.1 μg/m     TPA, 0.1 μg/ml MEZ or 0.25 μg/ml ADMB, in the presence or absence o     0.1 μg/ml H7.                                                              .sup.b Cell lysates were prepared and BCA 225 levels were determined by       doubledeterminant BCA 225 ELISA assay as described in "Detailed               Description of the Invention.                                                 .sup.c Membrane expression of BCA 225 was determined by flow cytometry        using a FACStar (Beckon Dickinson, Mountain View, CA) with CU18 monoclona     antibodies as described in "Detailed Description of the Invention." The       results are expressed as mean fluorescence intensity (MFI) units.             .sup.d The shedding of BCA 225 into the culture medium was calculated         using a doubledeterminant BCA 225 ELISA procedure as described in             "Detailed Description of the Invention.                                       .sup.e Values in brackets reflect the level of enhancement in BCA 225         relative to control (equivalent to 1.0).                                 

                  TABLE 3                                                         ______________________________________                                        Effect of the PKC Inhibitor H-7 on Upregulation of                            HLA Class II antigens and ICAM-1 by IFN-β and IFN-γ in T47D        Human Breast Carcinoma Cells.                                                 Experimental Antigenic Expression (MFI)                                       Conditions.sup.a                                                                           HLA Class II                                                                             ICAM-1                                                ______________________________________                                        Control      2,919      54,536                                                H-7           .sup. 3,490 (1.2).sup.b                                                                  54,080 (1.0)                                         IFN-β    31,413 (10.8)                                                                            97,013 (1.8)                                         IFN-β + H-7                                                                            32,190 (11.1)                                                                           108,902 (2.0)                                         IFN-γ  202,102 (69.2)                                                                           191,490 (3.5)                                         IFN-γ + H-7                                                                          210,812 (75.2)                                                                           188,544 (3.5)                                         ______________________________________                                         .sup.a T47D cells were incubated for 72 in the presence of 500 units/ml       IFNβ or 50 units/ml IFNγ, in the presence or absence of 1.0        μg/ml of H7. Cells were then incubated with Monoclonal Antibodies          specific for HLA Class II antigens or ICAM1 followed by fluorescinated        antimouse IgG antibody and then analyzed by flow cytometry with a Coulter     Epics IV FACS (Coulter Electronics, Hialeah, FL.) as described in             "Detailed Description of the Invention." Results are expressed as mean        fluorescence intensity (MFI) units.                                           .sup.b Values in brackets indicate the relative increase in expression        versus untreated controls (equivalent to 1.0).                           

                  TABLE 4                                                         ______________________________________                                        Effect of the PKC Inhibitor H-7 on the Enhanced                               Synthesis, Expression and Shedding of BCA 225 Induced by IFN-β           and IFN-γ in T47D Human Breast Carcinoma Cells.                                                           BCA 225                                                BCA 225     BCA 225    Shedding.sup.d                              Experimental                                                                             Synthesis.sup.b                                                                           Expression.sup.c                                                                         (ng/ml/10.sup.6                             Conditions.sup.a                                                                         (ng/mg/protein)                                                                           (MFI)      cells)                                      ______________________________________                                        Control    223         2,816      47                                          H-7        .sup. 230 (1.0).sup.e                                                                     2,560 (0.9)                                                                              38 (0.8)                                    IFN-β 374 (1.7)   5,716 (2.0)                                                                              69 (1.5)                                    IFN-β + H-7                                                                         361 (1.6)   5,714 (2.0)                                                                              66 (1.4)                                    IFN-γ                                                                              722 (3.2)   9,069 (3.2)                                                                              64 (1.4)                                    IPN-γ + H-7                                                                        694 (3.1)   8,171 (2.9)                                                                              64 (1.4)                                    ______________________________________                                         .sup.a T47D cells were incubated for 72 hr with 500 units/ml IFNβ or     50 units/ml of IFNγ, in the presence or absence of 1.0 μg/ml of      H7.                                                                           .sup.b BCA 225 synthesis was calculated by ELISA using cell lysates as        described in "Detailed Description of the Invention.                          .sup.c BCA 225 cell surface expression was measured by flow cytometry         using a FACStar (Beckon Dickinson, Mountain View, CA) as described in         "Detailed Description of the Invention." Results are expressed as mean        fluorescence intensity (MFI) units.                                           .sup.d Shedding of BCA 225 into the culture medium was measured by ELISA      as described in "Detailed Description of the Invention.                       .sup.e Values in brackets indicate the relative change in BCA 225 in          comparison with controls (equivalent to 1.0).                            

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What is claimed is:
 1. A method for the upregulation of expression ofcell antigens without inducing shedding in a cell which comprisesadministering to the cell an amount of a protein kinase C activatoreffective for the upregulation of expression of a cell antigen withoutinducing shedding of said antigen from the cell.
 2. The method of claim1, wherein the protein kinase C activator is a synthetic protein kinaseC activator.
 3. The method of claim 2, wherein the protein kinase Cactivator is 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol.4. The method of claim 1, wherein the cell antigen is a tumor associatedantigen.
 5. The method of claim 1, wherein the cell antigen is a cellsurface antigen.
 6. The method of claim 4, wherein the cell antigen is ahistocompatibility antigen.
 7. The method of claim 4, wherein the tumorof the tumor associated antigen is a breast carcinoma.
 8. The method ofclaim 4, wherein the tumor of the tumor associated antigen is a coloncarcinoma.
 9. The method of claim 4, wherein the tumor associatedantigen is BCA
 225. 10. The method of claim 4, wherein the tumorassociated antigen is c-erb B2.
 11. The method of claim 4, wherein thetumor associated antigen is carcinoembryonic antigen.
 12. The method ofclaim 5, wherein the cell surface antigen is intercellular adhesionmolecule-1.
 13. A method for decreasing tumor cell heterogeneity byupregulating the expression of an antigen without inducing shedding ofsaid antigen which comprises administering an effective amount of aprotein kinase C activator to cells to induce upregulation of expressionof an antigen without inducing shedding of said antigen.
 14. The methodof claim 13, wherein the protein kinase C activator is a syntheticprotein kinase C activator.
 15. The method of claim 14, wherein theprotein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol.
 16. Themethod of claim 13, wherein the antigen is a tumor associated antigen.17. The method of claim 13, wherein the antigen is a histocompatibilityantigen.
 18. The method of claim 13, wherein the antigen is a cellsurface antigen.
 19. The method of claim 16, wherein the tumor of thetumor associated antigen is a breast carcinoma.
 20. The method of claim16, wherein the tumor of the tumor associated antigen is a coloncarcinoma.
 21. The method of claim 16, wherein the tumor associatedantigen is BCA
 225. 22. The method of claim 16, wherein the tumorassociated antigen is carcinoembryonic antigen.
 23. The method of claim16, wherein the tumor associated antigen is c-erb B2.
 24. The method ofclaim 18, wherein the cell surface antigen is intercellular adhesionmolecule-1.